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Abstract

Miwi2 and Mili are involved in piRNA production and transposon silencing in fetal gonads. It is known that the loss of Miwi2 leads to a complete exhaustion of germline in male mice. Whether the absence of Mili results in the same phenotype, has not been established yet. In this study we show that Mili is also required for germ cell maintenance, but presents a much weaker phenotype than Miwi2 with only a partial loss of the germ line observed in aged mice. The difference between the two mutant phenotypes could be explained by the fact that in Mili deficient fetal testes a small amount of piRNAs is produced that can mount a residual defense against transposable elements as determined by bisulfite sequencing data. We have determined that piRNAs made in the absence of Mili make up a small pool of piRNAs that are also present in the wild type mice. Mili mutant piRNAs are mainly primary in nature and are most likely bound by Miwi2 since Miwi2 partially retains its nuclear localization in the absence of Mili in mouse fetal gonadocytes. In summary, a primary piRNA biogenesis pathways exist in mouse in the absence of Mili that contribute to both germ line reprogramming and maintenance.
Miwi2 and Dnmt3L are involved in de novo DNA methylation in fetal gonads. Miwi2 and Dnmt3L mutant mice exhibit a depletion of germline with age. Dnmt3L mutant mice completely lose germ cells within a two-month period, while Miwi2 mutant mice exhibit Sertoli-only phenotype at nine-month of age. Nonetheless, the phenotype of both reprogramming mutants indicates Miwi2 and Dnmt3L’s possible role in germline maintenance. In this study we have shown that the main role of Miwi2 and Dnmt3L in establishing and maintaining the population of undifferentiated spermatogonia precursor cell population in the adult is executed through their role in reprogramming fetal gonadocytes. We have observed that even though Miwi2- and Dnmt3L-deficient spermatogonial precursor cells exhibit transposon de-repression, no apparent loss these cells can be associated with DNA damage. We hypothesize that actively transcribed TE containing locus generates hybrid transcripts triggering abnormal gene expression program in spermatogonia stem cells, which leads to a great reduction in the numbers of actual GFRa1pos stem cells observed n Miwi2 and Dnmt3L mutant testis. Overall, this study shows the importance of germ cell reprograming in establishing a healthy, functional stem cell population in the adult tissue.